Systems to modulate nucleic acid expression are important for a wide variety of basic and applied biological research areas, including functional genomics, gene therapy, vaccination, animal models for human diseases and biopharmaceutical protein production. In these applications, expression of a nucleic acid(s) of interest is preferably controlled in a quantitative and temporal way. Several artificial gene expression systems that are regulated by non-toxic effector molecules in a dose-dependent and reversible manner are currently available. The Tet system, in which gene expression is stringently controlled by tetracycline (Tc) or its derivative doxycycline (dox), is the most widely-used regulatory circuit (Baron et al. 2000; Gossen et al. 2001; Berens et al. 2003). This system is based on the sequence-specific, high-affinity binding of the Escherichia coli Tet repressor protein (TetR) to the tet operator (tetO) DNA sequence. Tc or dox binds to TetR and triggers a conformational change that prevents the repressor protein from binding to tetO. Fusion of the VP16 activation domain of herpes simplex virus to TetR resulted in the transcriptional activator tTA, which induces nucleic acid expression from tetO-containing promoters (Ptet) in eukaryotic cells (Gossen et al. 1992). The presence of Tc or dox abolishes tTA-tetO interaction and switches off gene expression (Tet-off system). A tTA variant with four amino acid substitutions in the TetR moiety was identified, which exhibits a reverse phenotype (Gossen et al. 1995). This reverse tTA (called rtTA) binds to Ptet exclusively in the presence of dox, but not in its absence (Tet-on system). Both Tet systems are now widely applied to control nucleic acid expression in eukaryotes, including mammals, plants and insects (reviewed in (Gossen et al. 2001)). Because long-term exposure to effectors is often undesirable, the Tet-on system is preferred in applications in which nucleic acid expression is to be sustained in a switched-off state for long periods, or when rapid induction of nucleic acid expression is required.
Unfortunately, the amino acid substitutions in rtTA that confer the reverse phenotype also affect its binding affinity for effectors. As a consequence, rtTA has lost the ability to be activated by Tc and other Tc-like compounds, and it requires 100-fold more dox for maximal induction than that is needed for tTA inhibition. These characteristics severely limit the in vivo use of the Tet-on system. For example, to activate Tet-on controlled transgene expression in the rat brain, the animals have to be fed with high doses of dox that are nearly toxic (Baron et al. 1997). Therefore, the Tet-on system, particularly its effector-sensitivity, has to be improved.
Previously, the Tet system has been optimized by introduction of rationally designed mutations (Baron et al. 1997; Baron et al. 1999), and by directed evolution in which random mutagenesis of the components of the Tet system was followed by functional screening of the mutants in bacterial or yeast assay systems (Gossen et al. 1995; Urlinger et al. 2000). However, these approaches are labor intensive, and mutations selected in bacterial or yeast assay systems are not necessarily improvements in higher eukaryotes.
Another disadvantage of current rtTA systems is the risk of reduced dox-dependence after multiple rounds of replication. This problem, for instance, arises during vaccination applications where replication of at least part of a pathogen is under control of an rtTA system. In such vaccination applications, protection against the pathogen is acquired by controlled, inducible replication of the at least part of a pathogen, preferably during a restrained time span. If, however, the rtTA system loses its dox-dependence, the at least part of a pathogen will constitutively replicate, resulting in too much pathogenic nucleic acid and/or proteins, involving a safety problem. The same kind of problem arises during other applications involving repeated rounds of amplification of rtTA. It is therefore desired to improve the genetic stability of current rtTA systems.